CN1743009A - Medicine carrier pseudoglucoprotein nano particle of double-target anti-liver-fibrosis and its preparing method - Google Patents
Medicine carrier pseudoglucoprotein nano particle of double-target anti-liver-fibrosis and its preparing method Download PDFInfo
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Abstract
The present invention relates to double target anti-hepatofibrosis medicine carrier-pseudoglucoprotein nano particles, namely, 6-phospho-mannitose derivative albuminoid nano particles and its preparation method. Said invention provides its chemical structure general formula. The invented pseudoglucoprotein nano particles can be used as medicine carrier. Firstly, under the inchondriosis of phagocyte of reticuloendothelial system of liver and spleen the nano particles on which the anti-hepatofibrosis medicine is carried can be passively targeted into the liver, then, under the particles-intaking action of cell the nano particles can be fed into cell interior so as to make the anti-hepatofibrosis medicine fully produce action in the interior of hepatic stellate cell, HSC, so that the therapeutic effect of medicine can be greatly raised.
Description
Technical field
The present invention relates to medical technical field, is a kind of pharmaceutical carrier of dual-target anti-hepatic fibrosis---Neoglycoproteins nanoparticle and preparation method thereof.
Background technology
Cell surface has special receptor, utilizes ligands specific to combine with microparticle surfaces, makes microgranule guiding cell, can change the bio distribution of microgranule.This class part has strong affinity to receptor, comprises the cell surface marker thing, as sugar, external source clusterin etc.Researcher is used sugar derivatives and is modified microgranule, can cause the targeting to leukocyte, alveolar sac, hepatocyte etc.Can discern galactose as hepatocyte, lung/core cell, fibrocyte can be discerned the Man-6-P ester, and leukocyte can be discerned 6-amino-mannose, and mononuclear phagocyte can be discerned mannose, N-acetyl-glucosamine.Find hepatic stellate cell (hepatic stellate cell after deliberation, HSC) under various impairment factors, can be activated and breed, make extracellular matrix synthetic in a large number, directly cause the formation of hepatic fibrosis, Man-6-P/insulin like growth factor among the HSC (M6P/IGF II) receptor, it is more to distribute in activation HSC.There is the 10%-20%M6P/IGF II receptor can be approximately directly at cell surface expression, its expression in the hepatic fibrosis process still can further increase, the Rd of cell surface is strengthened, Beljaars etc. with M6P to human albumin (Human Serum Albumin, HSA) modify, obtain Noviose protein carrier (M6P-HSA), can be by the HSC internalization, under endocytosis, enter cell interior (Beljaars L, Molema G, Weert B, et al.Albumin modified with mannose 6-phosphate:a potential carrier for selectivedelivery of antifibrotic drugs to rat and human hepatic stellate cell.Hepatology, 1999,29:1486-1493.).The complex that M6P-HSA and medicine form can be used as effective transport vehicle that medicine arrives HSC, and anti-hepatic fibrosis medicines is played a role in HSC inside.But only limit to the in vitro study of glycosyl modified albumin medicinal composition so far, not seeing has the appropriate formulation report.
Summary of the invention
The object of the invention provides a kind of dual-target anti-hepatic fibrosis medicines carrier---Neoglycoproteins nanoparticle and preparation method thereof.
Because the nanoparticulate carriers system has unique targeting; slow controlled release characteristics and protection drug effect; albuminous material is safety non-toxic again; non-immunogenicity; biodegradable; good biocompatibility; therefore after the present invention carries out the saccharifying structural modification with albumin; get Noviose protein nano grain by the desolvation legal system; it promptly is the nano_scale particle of substrate with the Neoglycoproteins; to enter in the body behind its medicine carrying; utilize the phagocytosis of the macrophage of the reticuloendothelial system that liver spleen etc. locates to enrich; the Neoglycoproteins nanoparticle passive target that is loaded with anti-hepatic fibrosis medicines is transported to liver etc. to be located; utilize Man-6-P/insulin like growth factor among Neoglycoproteins nanoparticle surface specific part and the HSC (M6P/IGF II) receptor-specific combination again; with the drug main moving-target to being transported to HSC; realize the active and passive dual-target of anti-hepatic fibrosis medicines; improve curative effect of medication greatly, reduce drug side effect.
Neoglycoproteins nanoparticle skeleton symbol of the present invention is
M6P represents 6-phosphomannose derivant, and general formula is as follows:
Wherein, the R group is represented the substituent group on the aromatic ring, substituting group position can be positioned at the neighbour,, para-position, can not have replacement, the single replacement, also can be polysubstituted, substituent group is selected from:
(1)H、F、Cl、Br、I；
(2) methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group, sec-butyl, amyl group, tertiary pentyl, sec-amyl, isopentyl, amido, cyano group, nitro, methoxyl group, ethyoxyl, propoxyl group, butoxy, isobutoxy, tert-butoxy, sec-butoxy;
The M group is represented substituent group sulfo-carbonyl diamine or the azo group that is connected with albumin nano granular surface amino groups acid residue on the aromatic ring; Substituting group position can be positioned at the neighbour,, para-position, can singly replace, also can be polysubstituted;
The preferred 6-phosphoric acid-1-of M6P (4-sulfo-carbonyl diamine phenol)-α-D-mannopyranose, its R=H, M=4-sulfo-carbonyl diamine phenol;
N represents 1～60;
Albumin is selected from ovalbumin, lactalbumin, legumelin, serum albumin, and preferred bovine serum albumin (BovineSerum Albumin, BSA), the human albumin (Human Serum Albumin, HSA).
The preferred Neoglycoproteins nanoparticle of the present invention is 6-phosphoric acid-1-(4-sulfo-carbonyl diamine phenol)-α-D-mannopyranose albumin nano granular, preparation method is: by after paranitrophenol-α-D-mannopyranose phosphorylation again nitroreduction get para-aminophenol-6-phosphoric acid-α-D-mannopyranose (pap-M6P), activate back and albumin coupling with thiophosgene, get para-aminophenol-6-phosphoric acid-α-D-mannopyranose albumin, adopt the desolvation method more promptly.Reaction process is as follows:
Concrete steps are:
(1) five oxygen acetyl-β-the D-mannopyranose 1. in preparation
The D-mannose is at acetic anhydride: pyridine solution=8～12: 11～15, under-20 ℃～-5 ℃ conditions, react 4h, put again 0 ℃ 2 days, five oxygen acetyl-β-the D-mannopyranose 1.;
(2) acetyl-α-the D-mannopyranose 2. for preparation paranitrophenol-four oxygen
With paranitrophenol and five oxygen acetyl-β-D-mannopyranose 1. with ZnCl
2For catalyst in 160 ℃ the reaction 30min, paranitrophenol-four oxygen acetyl-α-the D-mannopyranose 2.;
(3) paranitrophenol-α-the D-mannopyranose 3. in preparation
2. paranitrophenol-four oxygen acetyl-α-D-pyrrole south mannose in absolute methanol, adds NaOCH
3Reflux, paranitrophenol-α-the D-mannopyranose 3.;
(4) phosphoric acid-paranitrophenol-α-the D-mannopyranose 4. for preparation 6-
Paranitrophenol-α-D-mannopyranose is 3. at pyridine: acetonitrile: water=5～15: 20～30: in 0～2 solution with POCl
3React to such an extent that 6-phosphoric acid-paranitrophenol-α-the D-mannopyranose 4.;
(5) phosphoric acid-para-aminophenol-α-the D-mannopyranose 5. for preparation 6-
6-phosphoric acid-paranitrophenol-α-D-mannopyranose is 4. at methanol: water=3～9: in 1～3 the mixed liquor, under the Pa-C catalytic action, with H
2React to such an extent that 6-phosphoric acid-para-aminophenol-α-the D-mannopyranose 5.;
(6) (4-isothiocyanate phenol)-α-the D-mannopyranose 6. for preparation 6-phosphoric acid-1-
6-phosphoric acid-para-aminophenol-α-D-mannopyranose is 5. in ethanol and water=5～15: in 1～3 the mixed liquor, with CSCl
2React 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-the D-mannopyranose 6.;
(7) preparation Neoglycoproteins [M6P] n-albumin 7.
6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose is aqueous solution 6., with bovine serum albumin or human albumin or other albuminous borate buffer solution room temperature reaction 18h, get Neoglycoproteins [M6P] n-albumin 7., 6. (1～800: 1) can get the different Neoglycoproteins of degree of glycosylation [M6P] n-albumin, n is 1～60 with albuminous ratio by adjusting 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose;
(8) preparation Neoglycoproteins nanoparticle 8.
7. Neoglycoproteins is mixed with aqueous solution or is dissolved in 10mMNaCl or the KCl neutral salt solution, control pH value 5-11, stirring state continues to add an amount of desolvation agent down, the desolvation agent is selected from dehydrant ethanol, methanol, acetone etc., after forming the Neoglycoproteins nanoparticle, add an amount of cross-linking agent glutaraldehyde or methyl polyethylene-dextran or stabilizing agent lactic acid, continuous stirring is impelled the nanoparticle crosslinking curing more than 12 hours, get Neoglycoproteins nanoparticle colloidal suspension, ℃ spent the night in these suspension slight fever to 20～37, lyophilizing gets final product after removing resolvating agent, gained nanoparticle particle diameter 50-300nm.
The preparation method of carrying anti-hepatic fibrosis medicine Neoglycoproteins nanoparticle is:
In Neoglycoproteins [M6P] n-albumin aqueous solution or neutral salt solution 10mM NaCl or KCl 7., the water or the alcoholic solution that add the anti-hepatic fibrosis medicine, the anti-hepatic fibrosis medicine is selected from sodium ferulate, cinobufotoxin, bufalin, colchicine, Malotilate, control pH value 5-11, stirring state continues to add an amount of desolvation agent down, the desolvation agent is selected from dehydrant ethanol, methanol, acetone etc., after forming medicine carrying Neoglycoproteins nanoparticle, add an amount of cross-linking agent glutaraldehyde or methyl polyethylene-dextran or stabilizing agent lactic acid, continuous stirring is impelled the nanoparticle crosslinking curing more than 12 hours, gained medicine carrying Neoglycoproteins nanoparticle colloidal suspension is spent the night in 20～37 ℃ of following slight fevers, remove lyophilizing behind the resolvating agent, particle diameter be the nanoparticle of 50-300nm.If add proper amount of surfactant poloxamer or lecithin etc. simultaneously, can improve nanoparticle envelop rate and carrying drug ratio in the preparation process with anti-hepatic fibrosis medicines.
The specific embodiment
Now in conjunction with the embodiments, the present invention is described in detail
Embodiment oxygen acetyl-β-D-mannopyranose preparation 1. in 1: five
In the 500ml round-bottomed flask, add acetic anhydride 200ml, pyridine 260ml, cryosel is bathed and is cooled to-15 ℃, adds D-mannose 24g in half an hour in batches, stirs 4h, the gained colorless cleared solution put 0 ℃ 2 days, jolting therebetween is several times.Slowly pour into after solution firmly shakes up in the 3L water that contains trash ice, stir 45min, crystallization is separated out, and filters, and filter cake washes after drying with water and spends the night, and gets 1. 40.3g of white powder five oxygen acetyl-β-D-mannopyranose, productive rate 77.5%.ESI-MS(positive-ion?mode)：413.02(M+Na
+，100％)。1H?NMR(D
2O)：1.94-2.17(m，15H，CH
3CO)，4.07-5.97(m，7H，C
1-6-H)。
Embodiment 2: paranitrophenol-four oxygen acetyl-α-D-mannopyranose preparation 2.
Add paranitrophenol 15g in the 500ml round-bottomed flask, five oxygen acetyl-β-D-mannopyranose is 15g 1., ZnCl
20.6g oil bath is heated to 160 ℃, stirs 30min, the black fused mass is cooled to 70 ℃, slowly adds benzole soln 240ml, and after the gained brown solution washed with water, it was almost colourless then to be washed till water layer with 1mol/l NaOH, washes anhydrous CaCl again with water
2Drying to crocus syrupy shape liquid, adds an amount of anhydrous alcohol solution in 35 ℃ of evaporated under reduced pressure, (35 ℃ of activated carbon decolorizings, 20min), be evaporated to crystal once more and separated out, the room temperature cooling is placed on-20 ℃ and spends the night, separate out a large amount of crystal filter white powder, be paranitrophenol-four oxygen acetyl-α-D-mannopyranose 2., mother solution can further concentrate recrystallization and get product 2., amounts to 11.3g, productive rate 62.8%, ([α]
20D+93 ° of c1, chloroform).Also activated carbon decolorizing gained solution can be used column chromatography separating purification (ethyl acetate-petroleum ether, 1: 2) after 30 ℃ of evaporated under reduced pressure, eluent gets white powder in 30 ℃ of drying under reduced pressure.(the thin layer chromatography developing solvent: ethyl acetate-petroleum ether, 1: 2, Rf=0.38).ESI-MS(positive-ionmode)：492.15(M+Na
+，100％)。1H?NMR(CDCl
3)：8.23(d，2H，J＝9.1Hz，Ar-H-meta)，7.21(d，2H，J＝9.1Hz，Ar-H-ortho)，2.11(m，12H，CH
3CO)，4.00-5.63(m，7H，C
1-6-H)。
Embodiment 3: paranitrophenol-α-D-mannopyranose preparation 3.
Add absolute methanol 96ml in the 250ml three-necked bottle, paranitrophenol-four oxygen acetyl-α-D-mannopyranose is 8g 2., stir suspension, add 0.2mol/l NaOCH
31.2ml, be heated to backflow 8min, to separate out in 30 ℃ of evaporated under reduced pressure to crystal, room temperature cooling is placed on-20 ℃, spends the night, and filters after drying and gets white powder, be i.e. 3. 3.5g of paranitrophenol-α-D-mannopyranose, productive rate 68.7%.([α]
20D+145 ° of c 0.2, water), (thin layer chromatography developing solvent: CHCl
3/ MeOH/H
2O, 13: 8: 2, Rf=0.86).ESI-MS(negtive-ion?mode)：346.05(M+HCOOH-1，100％)。1H?NMR(D
2O)：8.25(d，2H，J＝9Hz，Ar-H-meta)，7.28(d，2H，J＝9Hz，Ar-H-ortho)，5.77(s，1H，C
1-H)，3.61-4.19(m，6H，C
2-5，6a，6b-H)。
Embodiment 4:6-phosphoric acid-paranitrophenol-α-D-mannopyranose preparation 4.
In the 25ml round-bottomed flask, add pyridine 2.8ml, acetonitrile 7ml, water 0.28ml, the back that stirs adds 3. 2.1g of paranitrophenol-α-D-mannopyranose, slowly is added dropwise to POCl under the condition of ice bath
32.8ml, stirring 1.5h, the gained settled solution is poured into and 84g is housed on ice, and after the thawing, regulating pH with 2.5mol/l NaOH is 7.0, uses column chromatography separating purification (MeOH-DCM, 1: 1) (developing solvent: CHCl after 30 ℃ of evaporated under reduced pressure
3/ MeOH/H
2O, 13: 8: 2, Rf=0.29), eluent got white powder in 30 ℃ of drying under reduced pressure, i.e. 4. 1.85g of 6-phosphoric acid-paranitrophenol-α-D-mannopyranose, productive rate 69.5%.ESI-MS(negtive-ion?mode)：380.00(M-1，100％)。1H?NMR(D
2O)：8.12(d，2H，J＝8.7Hz，Ar-H-meta)，7.15(d，2H，J＝8.9Hz，Ar-H-ortho)，5.63(s，1H，C
1-H)，3.60-4.07(m，6H，C
2-5，6a，6b-H)。
Embodiment 5:6-phosphoric acid-para-aminophenol-α-D-mannopyranose preparation 5.
(6: 1, v/v), 6-phosphoric acid-paranitrophenol-α-D-mannopyranose is 0.7g 4., 10%Pa-C 0.075g, gained suspension room temperature, normal pressure, the down logical H of stirring for the mixed liquor of adding 49ml methanol and water in the 100ml three-necked bottle
2Stream 4h, thin layer chromatography (TLC) monitoring reaction.In 30 ℃ of evaporated under reduced pressure to 3-4ml, the adularescent crystal is separated out, sucking filtration is removed, and filtrate is concentrated into 2ml, adds an amount of methanol, a large amount of yellowish-brown solids are separated out, sucking filtration, filter cake is washed with methanol, and filtrate is continued evaporated under reduced pressure and is got brown solid, merging with dry cake before is 5. 0.55g of product 6-phosphoric acid-para-aminophenol-α-D-mannopyranose, productive rate 85.9%.(thin layer chromatography developing solvent: CHCl
3/ MeOH/H
2O, 13: 8: 2, Rf=0.14).ESI-MS(negtive-ion?mode)：350.00(M-1，100％)。1H?NMR(D
2O)：7.07(d，2H，J＝8.9Hz，Ar-H-meta)，7.02(d，2H，J＝8.9Hz，Ar-H-ortho)，5.48(s，1H，C
1-H)，4.12-3.49(m，6H，C
2-5，6a，6b-H)。
Embodiment 6:6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose preparation 6.
(9: 1, v/v), 6-phosphoric acid-para-aminophenol-α-D-mannopyranose is 0.217g (0.62mmol) 5., slowly is added dropwise to CSCl under the condition of ice bath for the mixed liquor of adding 50ml dehydrated alcohol and water in the 100ml round-bottomed flask
20.27ml (3.5mmol), stir 2h, uncovered logical N
2Stir 3h, transferring pH with 0.1mol/l NaOH under the condition of ice bath is 6.0, gets yellow solid in 30 ℃ of evaporated under reduced pressure, with column chromatography separating purification (CHCl
3/ MeOH/H
2O, 13: 8: 2) (developing solvent: CHCl
3/ MeOH/H
2O, 13: 8: 2, Rf=0.43), eluent got buff powder in 30 ℃ of evaporated under reduced pressure, i.e. 6. 0.225g of product 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose, productive rate 92.2% is in-20 ℃ of preservations.ESI-MS(negative-ion?mode)：392.03(M-1，100％)。1H?NMR(D
2O)：7.26(d，2H，Ar-H-meta)，7.10(d，2H，Ar-H-ortho)，5.54(s，1H，C
1-H)，3.45-4.09(m，6H，C
2-5，6a，6b-H)。
Embodiment 7: the preparation of Neoglycoproteins [M6P] n-BSA lyophilized powder
Get 6. 35.37mg (0.09mmol) of 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose, be dissolved in the 5ml distilled water, get sugar derivatives solution.In the 20ml round-bottomed flask, add BSA21.78mg (0.23 μ mol), borate buffer solution 7.5ml (pH 9.0), slowly add sugar derivatives solution again, stirring at room 18h, gained solution 0.15mol/lNaCl (1000ml), distilled water (2000ml) dialysis, the dialysis solution lyophilizing gets white powder, i.e. target product Neoglycoproteins [M6P]
3-BSA.Crude product is through sephadex G-25 column separating purification, and 0.15mol/l NaCl is an eluent, and lyophilizing promptly.
Embodiment 8: the preparation 8. of Neoglycoproteins nanoparticle
Precision takes by weighing 30mg Neoglycoproteins lyophilized powder, is dissolved in the 4.0ml distilled water, and pH value is 8.5, and the speed with 0.5ml/min under the stirring at room continues to add 7.0ml 95% ethanol, can form the Neoglycoproteins nanoparticle.Behind the desolvation, add 8% glutaraldehyde water solution, 30 μ l and make microgranule crosslinked, stir more than the 12h simultaneously in the cross-linking process.Gained Neoglycoproteins nanoparticle 8., particle diameter is 50-300nm.
The Neoglycoproteins lyophilized powder is dissolved among the neutral salt solution 10mMNaCl (or KCl), and method is the same, also can make the Neoglycoproteins nanoparticle, and particle diameter is 50-300nm.
Embodiment 9: the preparation of carrying anti-hepatic fibrosis medicine sodium ferulate Neoglycoproteins nanoparticle
7. precision takes by weighing 30mg Neoglycoproteins lyophilized powder, be dissolved in the 4.0ml distilled water, anti-hepatic fibrosis medicine sodium ferulate aqueous solution (or alcoholic solution) 2.0ml that adds 5mg/ml again, pH value is 8, speed with 0.5ml/min under the stirring at room continues to add 5.0ml 95% ethanol, can form sodium ferulate Neoglycoproteins nanoparticle.Behind the desolvation, add 8% glutaraldehyde water solution, 10 μ l and make microgranule crosslinked, stir more than the 12h simultaneously in the cross-linking process.Gained sodium ferulate Neoglycoproteins nanoparticle particle diameter is 50-300nm.
The Neoglycoproteins lyophilized powder is dissolved among the neutral salt solution 10mMNaCl (or KCl), and method also can make sodium ferulate Neoglycoproteins nanoparticle with embodiment 9, and particle diameter is 50-300nm.
The preparation method of other anti-hepatic fibrosis medicines such as cinobufotoxin, bufalin, colchicine, Malotilate Neoglycoproteins nanoparticle is with embodiment 9.
Neoglycoproteins 10～300mg and anti-hepatic fibrosis medicine 1～200mg are with＞1 arbitrary proportion in 4.0ml distilled water or neutral salt solution, adopt the method identical to be prepared with embodiment 9, can obtain anti-hepatic fibrosis medicine Neoglycoproteins nanoparticle equally, particle diameter is 50～300nm.
Common anti-hepatic fibrosis medicines preparation enters whole body distribution in the body, and dosage is just big, not only wastes medicine, and can cause toxicity.And the present invention has realized the dual-target administration, and medicine is concentrated in HSC, can reduce dosage, has not only given full play to the therapeutical effect of medicine, and has reduced poisonous side effect of medicine, and is significant to effective control of hepatic fibrosis.
Claims (6)
1. medicine carrier pseudoglucoprotein nano particle of double-target anti-liver-fibrosis
Wherein M6P represents 6-phosphomannose derivant, and general structure is as follows:
The R group is represented the substituent group on the aromatic ring, substituting group position can be positioned at the neighbour,, para-position, can not have replacement, the single replacement, also can be polysubstituted, substituent group is selected from:
(1)H、F、Cl、Br、I；
(2) methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group, sec-butyl, amyl group, tertiary pentyl, sec-amyl, isopentyl, amido, cyano group, nitro, methoxyl group, ethyoxyl, propoxyl group, butoxy, isobutoxy, tert-butoxy, sec-butoxy;
The M group is represented substituent group sulfo-carbonyl diamine or the azo group that is connected with albumin nano granular surface amino groups acid residue on the aromatic ring; Substituting group position can be positioned at the neighbour,, para-position, can singly replace, also can be polysubstituted;
N represents 1～60;
Albumin is selected from serum albumin, ovalbumin, lactalbumin, legumelin.
2. by the described Neoglycoproteins nanoparticle of claim 1, it is characterized in that R is H, M is a 4-sulfo-carbonyl diamine phenol, and albumin is human albumin or bovine serum albumin, and it is 6-phosphoric acid-1-(4-sulfo-carbonyl diamine phenol)-α-D-mannopyranose protein nano grain;
3. the preparation method of the described 6-phosphoric acid-1-of claim 2 (4-sulfo-carbonyl diamine phenol)-α-D-mannopyranose protein nano grain, synthetic route is as follows:
Concrete steps are:
(1) five oxygen acetyl-β-the D-mannopyranose 1. in preparation
The D-mannose is at acetic anhydride: pyridine solution=8～12: 11～15, under-20 ℃～-5 ℃ conditions, react 4h, put again 0 ℃ 2 days, five oxygen acetyl-β-the D-mannopyranose 1.;
(2) acetyl-α-the D-mannopyranose 2. for preparation paranitrophenol-four oxygen
With paranitrophenol and five oxygen acetyl-β-D-mannopyranose 1. with ZnCl
2For catalyst in 160 ℃ the reaction 30min, paranitrophenol-four oxygen acetyl-α-the D-mannopyranose 2.;
(3) paranitrophenol-α-the D-mannopyranose 3. in preparation
2. paranitrophenol-four oxygen acetyl-α-D-mannopyranose in absolute methanol, adds NaOCH
3Reflux,
Paranitrophenol-α-the D-mannopyranose 3.;
(4) phosphoric acid-paranitrophenol-α-the D-mannopyranose 4. for preparation 6-
Paranitrophenol-α-D-mannopyranose is 3. at pyridine: acetonitrile: water=5～15: 20～30: in 0～2 solution with POCl
3React to such an extent that 6-phosphoric acid-paranitrophenol-α-the D-mannopyranose 4.;
(5) phosphoric acid-para-aminophenol-α-the D-mannopyranose 5. for preparation 6-
6-phosphoric acid-paranitrophenol-α-D-mannopyranose is 4. at methanol: water=3～9: in 1～3 the mixed liquor, under the Pa-C catalytic action, with H
2React to such an extent that 6-phosphoric acid-para-aminophenol-α-the D-mannopyranose 5.;
(6) (4-isothiocyanate phenol)-α-the D-mannopyranose 6. for preparation 6-phosphoric acid-1-
6-phosphoric acid-para-aminophenol-α-D-mannopyranose is 5. in ethanol and water=5～15: in 1～3 the mixed liquor, with CSCl
2React 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-the D-mannopyranose 6.;
(7) preparation Neoglycoproteins [M6P] n-albumin 7.
6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose is aqueous solution 6., with bovine serum albumin or human albumin or other albuminous borate buffer solution room temperature reaction 18h, get Neoglycoproteins [M6P] n-albumin 7., by adjust 6-phosphoric acid-1-(4-isothiocyanate phenol)-α-D-mannopyranose 6. with albuminous ratio 1～800: 1, can get the different Neoglycoproteins of degree of glycosylation [M6P] n-albumin, n is 1～60;
(8) preparation Neoglycoproteins nanoparticle 8.
7. Neoglycoproteins is mixed with aqueous solution or is dissolved in 10mM NaCl or the KCl neutral salt solution, control pH value 5-11, stirring state continues to add an amount of desolvation agent down, the desolvation agent is selected from dehydrant ethanol, methanol, acetone, after forming the Neoglycoproteins nanoparticle, add an amount of cross-linking agent glutaraldehyde or methyl polyethylene-dextran or stabilizing agent lactic acid, continuous stirring is impelled the nanoparticle crosslinking curing more than 12 hours, get Neoglycoproteins nanoparticle colloidal suspension, ℃ spent the night in these suspension slight fever to 20～37, lyophilizing gets final product after removing resolvating agent, gained nanoparticle particle diameter 50-300nm.
4. the application in the preparation anti-hepatic fibrosis medicines by claim 1 or 2 or 3 described Neoglycoproteins nanoparticles.
5. carry anti-hepatic fibrosis medicines Neoglycoproteins nanoparticle preparation method, it is characterized in that 7. the described Neoglycoproteins of claim 3 [M6P] n-albumin is made into finite concentration aqueous solution or neutral salt solution, the aqueous solution or the alcoholic solution that add anti-hepatic fibrosis medicines, anti-hepatic fibrosis medicines is selected from sodium ferulate, cinobufotoxin, bufalin, colchicine, Malotilate, control pH value 5-11, stirring state continues to add an amount of desolvation agent down, the desolvation agent is selected from dehydrant ethanol, methanol, acetone, after forming medicine carrying Neoglycoproteins nanoparticle, add an amount of cross-linking agent glutaraldehyde or methyl polyethylene-dextran or stabilizing agent lactic acid, continuous stirring is impelled the nanoparticle crosslinking curing more than 12 hours, gained medicine carrying Neoglycoproteins nanoparticle colloidal suspension is spent the night in 20～37 ℃ of following slight fevers, remove resolvating agent after lyophilizing get final product.
6. by the described medicine carrying Neoglycoproteins of claim 5 nanoparticle preparation method, it is characterized in that when adding anti-hepatic fibrosis medicines, also adding proper amount of surfactant poloxamer or lecithin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102940621A (en) * | 2012-11-27 | 2013-02-27 | 桂林医学院 | Application of methyl ferulic acid in preparation of medicine for preventing and curing hepatic fibrosis |
| CN104725442A (en) * | 2015-04-15 | 2015-06-24 | 湘潭大学 | P-nitrophenyl-alpha-D-mannopyranoside preparation method |
| CN106432371A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannoprotein conjugates and preparation method and application thereof |
| US11498960B2 (en) | 2017-07-11 | 2022-11-15 | Alexion Pharmaceuticals, Inc. | Polypeptides that bind complement component C5 or serum albumin and fusion proteins thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018141678A1 (en) * | 2017-01-31 | 2018-08-09 | Medizinische Hochschule Hannover (Mhh) | Natural compounds and fibrosis |
-
2005
- 2005-09-20 CN CNB2005100297841A patent/CN100368022C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102940621A (en) * | 2012-11-27 | 2013-02-27 | 桂林医学院 | Application of methyl ferulic acid in preparation of medicine for preventing and curing hepatic fibrosis |
| CN102940621B (en) * | 2012-11-27 | 2014-08-13 | 桂林医学院 | Application of methyl ferulic acid in preparation of medicine for preventing and curing hepatic fibrosis |
| CN104725442A (en) * | 2015-04-15 | 2015-06-24 | 湘潭大学 | P-nitrophenyl-alpha-D-mannopyranoside preparation method |
| CN106432371A (en) * | 2016-10-31 | 2017-02-22 | 中国人民解放军第二军医大学 | Beta-1,2-D-oligomeric mannoprotein conjugates and preparation method and application thereof |
| US11498960B2 (en) | 2017-07-11 | 2022-11-15 | Alexion Pharmaceuticals, Inc. | Polypeptides that bind complement component C5 or serum albumin and fusion proteins thereof |
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